Maurizio Manno

Correlation between environmental and biological monitoring of exposure to benzene in petrochemical industry operators

The present work was aimed to study in petrochemical industry operators the correlation, if any, between environmental exposure to low levels of benzene and two biological exposure indexes in end-shift urine, i.e. trans, trans-muconic acid (t,t-MA) and S-phenylmercapturic acid (SPMA). Exposure to benzene was assessed in 133 male subjects employed in outdoor operations in a petrochemical plant, using personal passive-diffusive air samplers worn at the breathing zone; adsorbed benzene was determined by GC-FID analysis. S-PMA was determined by a new HPLCMS/MS method, after (quantitative) acidic hydrolysis of the cysteine conjugate precursor. t,t-MA was measured by an HPLC-UV method. Smoking habits were assessed by means of a self-administered questionnaire. Both environmental and biological monitoring data showed that benzene exposure of petrochemical industry operators was low (mean values were 0.014ppm, 101mug/g creat, and 2.8mug/g creat, for benzene, t,t-MA, and S-PMA, respectively) if compared with the ACGIH limits. Cigarette smoking was confirmed to be a strong confounding factor for the urinary excretion of both metabolites: statistically significant increases of t,t-MA and S-PMA levels were recorded in smokers when compared to non-smokers (p<0.0001). The best correlation found was that between exposure to benzene and S-PMA levels, particularly in non-smokers. This was partly due to the hydrolysis of the S-PMA precursor N-acetyl-S-(1,2-dihydro-2-hydroxyphenyl)-l-cysteine, a crucial step of the new analytical method used, which indeed reduced the variability of the results by means of an improved standardization of this critical preanalytical factor. A weaker correlation was found between exposure to benzene and t,t-MA, possibly explained by the fact that the latter is also a metabolite of sorbic acid, a common diet component. In summary, even at such low levels of exposure, urinary metabolites proved to be a useful tool for assessing individual occupational exposure to benzene, S-PMA appearing to be a more specific biomarker than t,t-MA, particularly in non-smokers.

Biomarkers of nanomaterial exposure and effect: current status

Abstract Recent advances in nanotechnology have induced a widespread production and application of nanomaterials. As a consequence, an increasing number of workers are expected to undergo exposure to these xenobiotics, while the possible hazards to their health remain not being completely understood. In this context, biological monitoring may play a key role not only to identify potential hazards from and to evaluate occupational exposure to nanomaterials, but also to detect their early biological effects to better assess and manage risks of exposure in respect of the health of workers. Therefore, the aim of this review is to provide a critical evaluation of potential biomarkers of nanomaterial exposure and effect investigated in human and animal studies. Concerning exposure biomarkers, internal dose of metallic or metal oxide nanoparticle exposure may be assessed measuring the elemental metallic content in blood or urine or other biological materials, whereas specific molecules may be carefully evaluated in target tissues as possible biomarkers of biologically effective dose. Oxidative stress biomarkers, such as 8-hydroxy-deoxy-guanosine, genotoxicity biomarkers, and inflammatory response indicators may also be useful, although not specific, as biomarkers of nanomaterial early adverse health effects. Finally, potential biomarkers from “omic” technologies appear to be quite innovative and greatly relevant, although mechanistic, ethical, and practical issues should all be resolved before their routine application in occupational settings could be implemented. Although all these findings are interesting, they point out the need for further research to identify and possibly validate sensitive and specific biomarkers of exposure and effect, suitable for future use in occupational biomonitoring programs. A valuable contribution may derive from the studies investigating the biological behavior of nanomaterials and the factors influencing their toxicokinetics and reactivity. In this context, the application of the most recent advances in analytical chemistry and biochemistry to the biological monitoring of nanomaterial exposure may be also useful to detect and define patterns and mechanisms of early nanospecific biochemical alterations.

In vivo CYP2E1 phenotyping as a new potential biomarker of occupational and experimental exposure to benzene.

Assessing CYP2E1 phenotype in vivo may be important to predict individual susceptibility to those chemicals, including benzene, which are metabolically activated by this isoenzyme. Chlorzoxazone (CHZ), a specific CYP2E1 substrate, is readily hydroxylated to 6-OH-chlorzoxazone (6-OH-CHZ) by liver CYP2E1 and the metabolic ratio 6-OH-CHZ/CHZ in serum (MR) is a specific and sensitive biomarker of CYP2E1 activity in vivo in humans. We used this MR as a potential biomarker of effect in benzene-treated rats and, also, in humans occupationally exposed to low levels of benzene. Male Sprague-Dawley rats (375-400g b.w.) were treated i.p. for 3 days with either a 0.5ml solution of benzene (5mmol/kg b.w.) in corn oil, or 0.5ml corn oil alone. Twenty-four hours after the last injection, a polyethylene glycol (PEG) solution of CHZ (20mg/kg b.w.) was injected i.p. in both treated and control animals. After 2, 5, 10, 15, 20, 30, 45, 60, 90, 120, 180, and 240min from injection, 0.2ml blood was taken from the tip tail and stored at -20 degrees C until analysis. A modified reverse phase HPLC method using a 5microm Ultrasphere C18 column equipped with a direct-connection ODS guard column, was used to measure CHZ and its metabolite 6-OH-CHZ in serum. No statistically significant difference in the MR was observed, at any sampling time, between benzene-treated and control rats. The concentration-versus-time area under the curve (AUC), however, was lower (p<0.05, Mann-Whitney test), whereas the systemic clearance was higher (p<0.05) in treated than in control rats. Eleven petrochemical workers occupationally exposed to low levels of airborne benzene (mean+/-SD, 25.0+/-24.4microg/m(3)) and 13 non-exposed controls from the same factory (mean+/-SD, 6.7+/-4.0microg/m(3)) signed an informed consent form and were administered 500mg CHZ p.o. Two hours later a venous blood sample was taken for CHZ and 6-OH-CHZ measurements. Despite exposed subjects showed significantly higher levels of t,t-MA and S-PMA, two biomarkers of exposure to benzene, than non-exposed workers, no difference in the MR mean values+/-SD was found between exposed (0.59+/-0.29) and non-exposed (0.57+/-0.23) subjects. So, benzene was found to modify CHZ disposition, but not CYP2E1 phenotype in benzene-treated rats, nor in workers exposed to benzene, probably due to the levels of exposure being too low.

Glutathione Transferases and Glutathionylated Hemoglobin in Workers Exposed to Low Doses of 1,3-Butadiene

We evaluated glutathione transferase (GST) activities and the levels of glutathionylated hemoglobin in the RBC of 42 workers exposed to 1,3-butadiene in a petrochemical plant, using 43 workers not exposed to 1,3-butadiene and 82 foresters as internal and external controls, respectively. Median 1,3-butadiene exposure levels were 1.5, 0.4, and 0.1 μg/m3 in 1,3-butadiene-exposed workers, in workers not directly exposed to 1,3-butadiene, and in foresters, respectively. In addition, we determined in the peripheral blood lymphocytes of the same individuals the presence of GST polymorphic genes GSTT1 and GSTM1 and the distribution of GSTP1 allelic variants. Comparing the mean values observed in petrochemical workers with those of control foresters, we found a marked decrease of GST enzymatic activity and a significant increase of glutathionylated hemoglobin in the petrochemical workers. A weak but significant negative correlation was found between levels of 1,3-butadiene exposure and GST activity, whereas a positive correlation was found between 1,3-butadiene exposure and glutathionylated hemoglobin. A negative correlation was also observed between GST activity and glutathionylated hemoglobin. No influence of confounders was observed. Using a multiple linear regression model, up to 50.6% and 41.9% of the variability observed in glutathionylated hemoglobin and GST activity, respectively, were explained by 1,3-butadiene exposure, working setting, and GSTT1 genotype. These results indicate that occupational exposure to 1,3-butadiene induces an oxidative stress that impairs the GST balance in RBC, and suggest that GST activity and glutathionylated hemoglobin could be recommended as promising biomarkers of effect in petrochemical workers. (Cancer Epidemiol Biomarkers Prev 2008;17(11):3004–12)

CYP2E1 phenotype in Mexican workers occupationally exposed to low levels of toluene

UNLABELLED CYP2E1, an inducible enzyme present in different human tissues, metabolizes several potentially toxic substances including many volatile organic compounds (VOCs). One indirect way to monitor exposure to VOCs may be, therefore, the assessment of CYP2E1 activity in vivo using the chlorzoxazone (CHZ) test. GOAL To compare CYP2E1 activity in two groups of workers: one with a known occupational exposure to VOCs (exposed group) and the other employed in administrative tasks at two universities (control group) from the city of León, Guanajuato, México. MATERIAL AND METHODS (1) Passive diffusion monitors were used to evaluate individual levels of exposure to toluene, benzene and ethylbenzene in 48 persons (24 tannery workers and 24 administrative controls) during a 8h work shift; (2) after 12h fasting 500mg CHZ, a selective probe for assessing CYP2E1 activity, was orally administered and, after 2h, a venous blood sample was collected for HPLC plasmatic quantitative determination of CHZ and its mean metabolite 6-hydroxychlorzoxazone. RESULTS Toluene mean exposure levels were higher in the exposed group (2.86±2ppm vs. 0.05±0.005ppm; p<0.001). Also, in this group CYP2E1 activity was lower (p<0.05) and it decreased as the accumulated months of labor exposure increased (negative correlation, p<0.05). These results are in line with previous findings obtained from shoemakers exposed to various solvents but, interestingly, they are partly in contrast with those of another study in printers. CONCLUSION In spite of the relatively low levels of toluene exposure found for tannery workers, an effect on CYP2E1 activity was evident. Although the mechanism of this interaction is still unknown, the decrease in CYP2E1 activity per se might represent a health risk, considering that these workers may be less protected against other CYP2E1 substrates present in the labor setting or derived from an intentional exposure.

An Integrated Approach for the Environmental Characterization of a Wide Potentially Contaminated Area in Southern Italy

This paper deals with the environmental characterization of a large and densely populated area, with a poor reputation for contamination, considering the contribution of environmental features (air, soil, soil hydraulic and groundwater) and the potential effects on human health. The use of Geographic Information System (GIS) has made possible a georeferenced inventory and, by overlaying environmental information, an operational synthesis of comprehensive environmental conditions. The cumulative effects on environmental features were evaluated, taking into account superposition effects, by means of the Spatial MultiCriteria Decision Analysis (S-MCDA). The application of the S-MCDA for converging the combination of heterogeneous factors, related to soil, land and water, deeply studied by heterogeneous groups of experts, constitutes the novelty of the paper. The results confirmed an overall higher potential of exposure to contaminants in the environment and higher mortality rates in the study area for some tumours, but hospital admissions for tumours were generally similar to the regional trend. Besides, mortality data may be strictly dependent on the poor socioeconomic conditions, quality of therapy and a lack of welfare in the area relative to the rest of Italy. Finally, as regards the possible relationship between presence of contaminants in the environment and health conditions of the population no definite conclusions can be drawn, although the present study encourages the use of the new proposed methods, that increase the possibilities for studying the combined effect of more environmental factors.

Kidney and liver biomarkers in female dry-cleaning workers exposed to perchloroethylene

Blood and urine perchloroethylene and urine trichloroacetic acid, as markers of exposure, and serum AST, ALT, GGT and creatinine, urine total solutes and proteins, angiotensin converting enzyme, N-acetyl-ß-D-glucosaminidase and glutamine synthetase, as markers of effect, were measured in 40 dry-cleaning and 45 ironing-shop female workers. Average perchloroethylene air level in the dry-cleaning shops was 59.7 mg m-3, i.e. three-fold lower than the current A.C.G.I.H. TLV-TWA (170 mg m-3). No statistically significant difference in the mean values of any of the effect markers was observed between the two groups, except for AST which was significantly higher in drycleaners. In addition, a statistically significant correlation was observed in dry-cleaners between environmental perchloroethylene and total urinary solutes (r = 0.308, p < 0.05) or urine glutamine synthetase (r= 0.469, p < 0 .01), between glutamine synthetase and blood perchloroethylene in post-shift (r= 0.406, p < 0.01) or urinary perchloroethylene in post(r= 0.571, p < 0.001) or pre-shift (r= 0.586, p < 0.001), and between urinary perchloroethylene in pre-shift and GGT (r= 0.407, p < 0.05). Interestingly, some statistically significant correlations between exposure and effect indices were found in ironing-shop workers alone, as in all subjects. Finally, transaminases, GGT and total urinary proteins were influenced by age and alcohol consumption which were significantly higher in dry-cleaners, thus providing an explanation for some of the correlations observed. In conclusion, our results show a dose-related increase of glutamine synthetase activity,a marker of damage of the pars recta of the kidney proximal tubule, in the urine of female subjects exposed to perchloroethylene concentrations in the work environment lower than current A.C.G.I.H. TLV-TWA.Blood and urine perchloroethylene and urine trichloroacetic acid, as markers of exposure, and serum AST, ALT, GGT and creatinine, urine total solutes and proteins, angiotensin converting enzyme, N-acetyl-ß-D-glucosaminidase and glutamine synthetase, as markers of effect, were measured in 40 dry-cleaning and 45 ironing-shop female workers. Average perchloroethylene air level in the dry-cleaning shops was 59.7 mg m(-3), i.e. three-fold lower than the current A.C.G.I.H. TLV-TWA (170 mg (m-3)). No statistically significant difference in the mean values of any of the effect markers was observed between the two groups, except for AST which was significantly higher in drycleaners. In addition, a statistically significant correlation was observed in dry-cleaners between environmental perchloroethylene and total urinary solutes (r = 0.308, p < 0.05) or urine glutamine synthetase (r= 0.469, p < 0 .01), between glutamine synthetase and blood perchloroethylene in post-shift (r= 0.406, p < 0.01) or urinary perchloroethylene in post(r= 0.571, p < 0.001) or pre-shift (r= 0.586, p < 0.001), and between urinary perchloroethylene in pre-shift and GGT (r= 0.407, p < 0.05). Interestingly, some statistically significant correlations between exposure and effect indices were found in ironing-shop workers alone, as in all subjects. Finally, transaminases, GGT and total urinary proteins were influenced by age and alcohol consumption which were significantly higher in dry-cleaners, thus providing an explanation for some of the correlations observed. In conclusion, our results show a dose-related increase of glutamine synthetase activity,a marker of damage of the pars recta of the kidney proximal tubule, in the urine of female subjects exposed to perchloroethylene concentrations in the work environment lower than current A.C.G.I.H. TLV-TWA.

Ethics in biomonitoring for occupational health

Biological monitoring, i.e., the use of biomarkers for the measurement of systemic human exposure, effects and susceptibility to chemicals has increased considerably in recent years. Biomonitoring techniques, originally limited to a few metals and other chemicals in the workplace, are currently applied to a large number of exposure situations and have become a useful tool for occupational and environmental health risk assessment. Almost any biomonitoring program, however, entails a number of relevant ethical issues, which concern all the phases of the entire process, from the selection of the biomarker to the study design, from the collection, storage and analysis of the biological sample to the interpretation, communication and management of the results, from the (truly?) informed consent of the worker to the independence and autonomy of the occupational health professional. These issues require a balanced assessment of the interests and responsibilities of all the parties, the worker primarily, but also the employer, the occupational health professional, the health authorities and, for research studies on new biomarkers, also the scientists involved. Ideally, decisions of ethical relevance concerning biomarkers should be based on, and respectful of the best scientific, legal and ethical evidence available. When, however, a conflict should arise, before any decision is taken a thorough risk-benefit analysis should be done, at the beginning of the process and after listening to the workers and the management involved, by the occupational physician or scientist, based on his/her professional experience, independent judgement and individual responsibility.

Comparison of hydrolysis and HPLC/MS/MS procedure with ELISA assay for the determination of S-phenylmercapturic acid as a biomarker of benzene exposure in human urine.

The present study compared three methods for the determination of S-phenylmercapturic acid (S-PMA), a metabolite of benzene, in human urine: a HPLC/MS/MS technique with two different sample treatments (strong and partial hydrolysis) and a commercial assay based on anti-S-PMA monoclonal antibodies with chemiluminescence detection. Biological monitoring was done on 126 volunteers and the results were compared for the three methods and also with benzene exposure levels (range <3.0-592.5 μg/m(3)). The correlation between environmental monitoring data and S-PMA levels in non-smokers (n=73) was highly significant (p<0.0001, Students t-test) for both HPLC/MS/MS methods (r=0.65 both for strong acidic hydrolysis of the urine and hydrolysis at pH 2) but not for the immunoassay, which overestimated the S-PMA levels by about 8 μg/g creatinine (creat.). Therefore the immunoassay is only useful as a semiquantitative screening test, but quantitative results need to be confirmed by a more accurate method like HPLC/MS/MS. The HPLC/MS/MS procedure with strong acid hydrolysis led to a recovery of S-PMA about double that using pH 2 hydrolysis, giving more accurate results. The difference between the results with the two methods makes it difficult to compare the strong acidic hydrolysis data with the ACGIH BEI value of 25 μg/g creat. since the BEI(®) documentation is based on data collected in pH conditions that were not always controlled, which may underestimate the true S-PMA concentration. Besides, as levels of benzene exposure were high, smoking was not considered a confounding factor. The BEI for S-PMA in end of shift urine samples could be reconsidered when sufficient data are available from studies where the analyses are carried out in comparable conditions of hydrolysis and monitoring only non-smoking subjects.

Effects of tricresylphosphate on esterase activity of rat serum and tissues.

The effect of tricresylphosphate (TCP) was studied in vitro and in vivo on the rat liver and brain enzymes acetylcholinesterase (ACC), butyrylcholinesterase (CHE), arylesterase (ARE), aliesterase (ALI), and the microsomal nicotinamide-adenine dinucleotide phosphate oxidase (NADPH2-oxidase) system. The results show that, in the male rat, TCP given intraperitoneally induces an increase in liver microsomal ARE AND NADPH2-oxidase and a decrease in ALI and cholinesterase; no activation of ARE and NADPH2-oxidase is observed in female rats.